Mycobacteria species, exemplified by M. tuberculosis and M. leprae are the cause of major diseases in humans. In this R21 `high risk/high impact'application we propose an exploratory program to define early steps in the biosynthesis of phosphatidylinositol mannosides (PIMs). PIMs are a family of mannolipids that plays a critical role in the pathogenesis of mycobacteria, because they are biosynthetic precursors in the assembly of the key cell surface glycoconjugates lipomannan (LM) and lipoarabinomannan (LAM). PIM biosynthesis occurs in the cytoplasmic membrane and there is considerable evidence to suggest that it occurs by the sequential addition of mannose residues to PI. We hypothesize that the first three mannosyltransfer reactions of PIM biosynthesis occur on the cytoplasmic face of the membrane and use GDP-mannose as the mannosyl donor;these reactions are catalyzed by the mannosyltransferases (ManTs) PimA, PimB and an undefined enzyme that we term PimC*. We further propose that subsequent mannosylation reactions use polyprenol-phosphate-mannose (PPM) as the mannosyl donor and occur on the membrane's periplasmic face. The enzyme that catalyzes the first of these periplasmically oriented reactions is unknown;we refer to it as PimD*. To accommodate the topological split in PIM biosynthesis, we propose that the trimannosylated glycolipid (AcPIM3) produced by PimC* must flip across the membrane in order to be further mannosylated. Since non-catalyzed phospholipid flipping is a rare event, we propose that AcPIM3 flipping requires a lipid translocator or flippase (referred to herein as Flp*), a protein that remains to be identified. The nexus of three steps surrounding the topological split in the PIM assembly pathway thus remains to be defined. In this "proof of principal" project evolving from close interactions between the three principal investigators over the last year, we propose two specific aims to identify the protein components required for these steps. Our studies will exploit the experimental model Mycobacterium smegmatis in conjunction with a multi-faceted approach including bioinformatics, mutational analysis, and biochemical reconstitution of transport activity. Our aims are: 1) To identify PimC* and PimD*, the ManTs that catalyze the mannosyltransfer reactions that occur immediately prior to and after the transbilayer translocation of a PIM intermediate. 2) To develop a reconstitution system that recapitulates Flp*-catalyzed PIM flip- flop and to use this system to identify Flp*, the flippase responsible for flipping AcPIM3 across the membrane. Thus the unifying theme of this proposal encompasses filling in `missing steps'in the biogenesis of PIMs, establish OUR hypothesized pathway, all in relation to identifying unique, extracellular targets. PUBLIC HEALTH RELEVANCE: Herein, two exploratory specific aims are proposed to address the vital missing gaps in the assembly of core glycolipids in Mycobacterium.